My laboratory has devised in vitro selection and directed evolution methods for the isolation of RNA, DNA and protein molecules with specific binding or catalytic properties. We have evolved new ribozymes that catalyze a wide range of different reactions, supporting the idea that ribozymes could have played an important role in early cells, prior to the evolution of protein synthesis and protein enzymes. We are now attempting to evolve an RNA replicase - an RNA molecule that is a good enough RNA polymerase to copy its own sequence. Such molecules are thought to have been key intermediates in the origin of life. We have been able to select for new ribozymes that can catalyze ligation reactions with activated oligonucleotides, and one of these new ribozymes is able to use nucleoside triphosphates in polymerase-like reactions. These ribozymes provide exciting starting points for the evolution of replicase molecules. In addition, the structures of several aptamers and ribozymes are being examined in collaborative projects with crystallography and NMR labs.
We have recently developed an in vitro selection and evolution system
that is applicable to proteins. Our approach is based upon the formation
of covalent protein-RNA conjugates during in vitro translation, as a result
of transfer of the nascent peptide to a puromycin residue attached to the
synthetic mRNA. This allows the protein moiety to be selected for binding
or catalytic function, while allowing the information to be retrieved by
RT-PCR of the attached mRNA. We have made very large protein libraries
of random, patterned and framework based sequences, and we are currently
selecting for novel binding proteins and enzymes. We are also exploring
the application of RNA-protein fusions to problems in functional genomics.
Selected Publications:
Ekland EH, Szostak JW, Bartel DP. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 1995; 269: 364-370.
Lohse P, Szostak JW. Ribozyme-catalysed amino acid transfer reactions. Nature 1996; 381: 442-444.
Roberts RW, Szostak JW. RNA-peptide fusions for the in Vitro
selection of peptides and proteins. Proc. Natl. Acad. Sci. USA 1997;
94: 12297-12302.